This proposal is devoted to the mechanisms of inherent genome instability, which occurs without external DNA damage and leads to numerous hereditary diseases in humans, as well as to genome rearrangements in cancer. Specifically, we will concentrate on genome instability caused by transcription-replication collisions, or mediated by interstitial DNA microsatellites. We have previously found that collisions between replication and transcription result in replication fork stalling and consequent genome instability. They are particularly profound in two situations: (i) head-on collisions between the replication fork and RNA polymerase, and (ii) co-directional collisions of the fork with R-loops formed at G-rich repeats upstream of the RNA polymerase. We propose to study the effects of these collisions on mutagenesis and gross-chromosomal rearrangements in bacterial and yeast experimental systems. We will determine the rates and spectra of mutations resulting from transcription-replication collisions in both organisms using new systems for forward mutagenesis assay combined with whole genome sequencing. To unravel the genetic controls of the collision-mediated genetic instabilities, we will carry out candidate gene analysis with an emphasis on genes that encode proteins implicated in transcription-couple repair, transcription-associated mutagenesis, R-loop processing, replication fork stabilization, replication fork restart, post-replication repair, and double-strand break repair. Our lab was the first to prove that DNA microsatellites can stall the progression of DNA replication, which is believed to be central for their ability to expand and induce chromosomal rearrangements leading to human disease. Here we concentrate on a particular type of microsatellite, interstitial telomeric sequences (ITSs). In humans, ITSs are polymorphic in length and were shown to co-localize with chromosomal fragile sites, recombinational hot spots, and sites of gross chromosomal rearrangements, some which were implicated in cancer and hereditary diseases. Surprisingly little is known, however, on the mechanisms that are responsible for the ITS-mediated genome instability. To bridge this gap, we propose to study the role of yeast and human ITS in genome instability in a yeast experimental system. Our preliminary data indicate that these ITSs can block the replication fork progression, expand or contract, induce mutagenesis at a distance as well as trigger gross chromosomal rearrangements. We will analyze the rates and spectra of these mutational events in a specifically designed yeast forward selection system. We will carry out candidate gene analysis and genome-wide screening to unravel genetic controls of ITS-mediated instability. We will study the details of replication and transcription through various ITSs using direct biochemical approaches established in the lab. Finally, we will analyze whether different types of genome instability triggered by the presence of ITSs could result from the formation of double-strand DNA breaks within these repeats.